An improved HPLC method for the quantitation of 3'-phosphoadenosine 5'-phosphate (PAP) to assay sulfotransferase...

Sulfotransferase-catalyzed (SULT-catalyzed) sulfation is responsible for hormone regulation and xenobiotic detoxification. In the current study, a sensitive and widely applicable method of reversed-phase HPLC-UV was developed for the determination of 3'-phosphoadenosine 5'-phosphate (PAP), a common product of different subtypes of sulfation reactions, in HepG2 cells. The analyte was separated on a ZORBAX Extend-C18 column with the mobile phase of methanol and water containing 75 mM KH(2)PO(4), 100mM NH(4)Cl, and 1mM 1-octylamine (pH 4.55), at a flow rate of 1.0 ml/min. The assay exhibited linearity over the range of 0.1-20 μM for PAP with a correlation coefficient of 0.9995. The total time per run was under 10 min. The intra- and inter-day precision was less than 7.2%, with accuracy in the range 82.6-102.0%. The method was applied to the determination of kinetic parameters K(m), V(m), and K(cat), of three different SULTs (SULT1A1, SULT2A1, and SULT1E1) in HepG2. This universal HPLC-UV method was easy to perform, economically feasible, and suitably efficient for the investigation of the enzyme kinetics of the SULT family using multiplex substrates.